Exposure to lead, especially of children, is of major concern in the US. Yet, determinants of sensitivity to the cytotoxic action of lead are unknown. The main goal of this project is to delineate cellular mechanisms of lead resistance. We propose to use rat C6 glioma cells as a model for astroglial cells, the cells that appear to protect neurons from lead toxicity. Twenty lead-resistant C6 sublines have been isolated, three have been characterized as to cross-resistance to other metals, and one has been demonstrated to show dominance to wild type cells in cell-cell hybrids. It is our hypothesis that transport and sequestration of lead are crucial determinants in lead toxicity and that lead-resistant variants will have genetic changes leading to up-regulation of proteins involved in these functions. The Specific Aims are: 1) To characterize lead-resistant variant C6 cells as to cross-resistance to other metals, lead transport and efflux, and dominance of the lead-resistant phenotype. 2) Genes (cDNA's) involved in lead resistance will be isolated by 2 strategies, one based on Differential Display and the second by expression cloning. Sequences of all cDNA's will be analyzed. Novel cDNA's will be used as probes to determine their expression levels in wild type and lead-resistant C6 cells. 3) Novel proteins will be expressed in E. coli, and antibodies to these proteins will be isolated. To gain insight into the function of these proteins, these antibodies will be used to study tissue distribution and sub-cellular localization. The abilities of these proteins to bind lead will also be determined, as it is likely that one or more novel proteins found will play a role in lead sequestration or transport. This work will help identify those proteins that play a role in lead toxicity and may lead to insight on individual susceptibility, increase our understanding of lead toxicology and may reveal new exposure markers and suggest new therapeutic strategies. An additional benefit results from our finding that lead-resistant cells are often cross-resistant to some other metals, so delineating the mechanisms of resistance should yield interesting information about the toxicology of other metals as well.